3,3′,5,5′-tetramethylbenzidine as electrochemical substrate for horseradish peroxidase based enzyme immunoassays.: A comparative study

The use of 3,3',5,5'-tetramethylbenzidine (TMB) as an electrochemical substrate for horseradish peroxidase (HRP) was investigated. HRP activity has been detected using flow injection analysis at a glassy carbon working electrode polarised at +100 mV versus Ag/AgCl in 0.1 mol(-1) citrate-phosphate buffer (pH 5.0). The optimum concentrations were 2 x 10(-4) mol l(-1) TMB and 10(-3) mol l(-1) H2O2. The detection limit obtained after 15 min of incubation was 8.5 x 10(-14) mol l(-1) HRP with the amperometric method. This limit was lower than that obtained using hydroquinone as HRP substrate and comparable to that with the p-aminophenyl phosphate-alkaline phosphatase system. Better performance was achieved with amperometric than spectrophotometric detection using TMB in a competitive ELISA for rabbit immunoglobulin G as a model analyte.